Various imaging systems and techniques have been used for biomedical applications, such as tissue mapping. One approach to tissue mapping involves multispectral imaging, where the tissue to be mapped is conjugate with a detector array. The array may be a charge coupled device (CCD) or a photo-diode array. In the array, the spatial data is directly obtained in the image. A tunable filter can be placed before the detector array, and images collected for each color setting of the filter. Liquid crystal tunable filters (LCTF) and acousto-optic tunable filters (AOTF) have been used and are available for such systems. See, for example, “Development of a Multi-Spectral Imaging System for Optical Diagnosis of Malignant Tissues” by Noordmans et al, Photonics West, 2005. See, also, “AOTF Microscope for Imaging with Increased Speed and Spectral Versatility”, by D. L. Farkas et al, published in Biophysical Journal, Volume 73, 1997.
As an alternative to a tunable filter, a volume hologram can be placed before the detector array, with images collected by pixel arrays positioned where each color is diffracted. See “Computed Tomography Based Spectral Imaging for Fluorescence Microscopy”, by M. R. Descour, Biophysical Journal, Volume 80, 2001.
However, each of these methods is limited in terms of temporal response time. The LCTF tuning method requires about 100 milliseconds per wavelength, which corresponds to about three 3 seconds per spectrum of 30 colors. The AOTF tuning method requires about 25 microseconds per wavelength, or 0.75 ms per spectrum of 30 colors. Both filters face CCD read-out times of at least 3 milliseconds per image or 90 milliseconds per datacube (i.e. x by y by wavelength arrays of data) containing 30 images. Integration times often necessitate much longer acquisition times, often as long as several seconds, which are too long for live biological samples that move on shorter time scales. The volume holographic method has no tuning time requirement, but is also limited by the CCD read-out rates and integration times. The AOTF imaging method represents the state of art in this field but faces limited throughput and spatial mis-registrations of one color with respect to another. Examples include U.S. Pat. No. 5,216,484, entitled “Real Time Imaging Spectrometer” and U.S. Pat. No. 5,796,512, entitled “Sub-Micron Imaging System Having an Acousto-Optical Tunable Filter”.
Another approach uses confocal microscopy. Confocal microscopy is a technique that creates high resolution images of a variety of objects. Confocal microscopy differs from conventional optical microscopy in that it uses a condenser lens to focus illuminating light from a point source into a very small, diffraction limited spot within a specimen, and an objective lens to focus the light emitted from that spot onto a small pinhole in an opaque screen. A detector which is capable of quantifying how much light passes through the pinhole at any instant is located behind the screen. Because only light from within the illuminated spot is properly focused to pass through the pinhole and reach the detector, any stray light from structures above, below, or to the side of the illuminated spot are filtered out. The image quality is therefore greatly enhanced as compared to other approaches.
A coherent image is built up by scanning point by point over the desired field of view and recording the intensity of the light emitted from each spot, as small spots are illuminated at any one time. Scanning can be accomplished in several ways, including for example, via laser scanning.
A particular field of study, known as multi-spectral confocal mapping, holds great promise for imaging cancer cells at the cellular and molecular levels. See “In-Vivo Cytometry: A Spectrum of Possibilities,” by D. L. Farkas et al, Cytometry, 2005. The National Institute of Health (NIH) has also encouraged research and development pertaining to in-vivo image guided cancer interventions. Biomedical researchers are working to detect tumor angiogenesis (via specific proteins and gene patterns), perform optical biopsies, perform treatments (optical surgery or photodynamic therapy), and monitor long term results. These goals require high spatial, spectral, and temporal resolution. Confocal microscopy can map millimeter sized fields of view with micron spatial resolution. Spectroscopic tools can provide multispectral data during laser excitation of fluorescence, 2-photon, and Raman spectroscopies. This is especially important when samples are labeled with a variety of molecular-specific contrast agents. See “Optical Molecular Imaging for Early Detection of Cancer,” by R. Richards-Kortum et al, OSA Topical Meeting on Biomedical Optics, April, 2004. Other applications include neural imaging, intra-cellular proteomics, micro-vascular testing, plaque detection, foodstuff testing, and the evaluation of pharmaceutical products. Further, these spatial and spectral measurements should allow for the detection of sub-millisecond dynamics in living systems. However, the above-described features and characteristics are not available in one cost-effective instrument.
Confocal microscopes are commercially available through entities such as Carl Zeiss, Nikon, and Olympus. Confocal endoscopes have also been employed in the medical field for endoscopic operations. Some of these endoscopes are quite advanced and reference is made to U.S. Pat. No. 6,522,444 entitled “Integrated Angled-Dual-Axis Confocal Scanning Endoscopes” by Michael J. Mandella, et al., dated Feb. 18, 2003 and assigned to Optical Biopsy Technologies Inc., for example. In this patent, there is described an integrated angled dual access confocal scanning endoscope which uses silicon micro-machined mirrors and fiber coupled instruments to enable better resolution and faster scanning. It employs one of two illumination beams in an angled dual access scanning endoscope which provides an assortment of reflected and fluorescent images. Endoscopes used for biological and medical imaging applications are further described in “MEMS Optical Scanners for Microscopes”, Miyajima et al, IEEE Journal of Selected Topics in Quantum Electronics, Volume 10, May/June 2004. In the described instruments, only one or a small number of wavelengths can be measured during each spatial scan. A high resolution fast multi-spectral confocal mapping technique and apparatus is desired.